Inducing cathepsin L expression/production, lysosomal activation, and autophagy of human dental pulp cells by dentin bonding agents, camphorquinone and BisGMA and the related mechanisms.

Camphorquinone (CQ) and resin monomers are included in dentin bonding agents (DBAs) and composite resin to restore tooth defects due to abrasion, crown fracture, or dental caries. DBAs, CQ, and bisphenol A-glycidyl methacrylate (BisGMA) applications influence the biological activities of the dental pulp. The current investigation aimed to delineate the effect of DBAs, CQ, and BisGMA on cathepsin L production/expression, lysosomal activity, and autophagy induction in human dental pulp cells (HDPCs). HDPCs were exposed to DBAs, CQ, or BisGMA with/without inhibitors for 24 h. Enzyme-linked immunosorbent assay was employed to determine the cathepsin L level in culture medium. The cell layer was utilized to measure cell viability by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl -tetrazolium bromide (MTT) assay. Real-time PCR was used to evaluate the mRNA expression. Western blotting or immunofluorescent staining was used to study protein expression. Lysosomal density was evaluated by lysotracker red staining. We found that DBAs, CQ, and BisGMA stimulated cathepsin L mRNA, protein expression, and production in HDPCs. In addition, CQ and BisGMA induced lysosomal activity, Beclin1, ATG12, LC3B, Bax, and p53 expression in HDPCs, indicating the stimulation of autophagy. Glutathione (GSH) prevented CQ- and BisGMA-induced cytotoxicity. Moreover, E64d, cathepsin L inhibitor (two cathepsin inhibitors), and Pifithrin-α (a p53 inhibitor) showed little preventive effect toward CQ- and BisGMA-induced cytotoxicity. Autophagy inhibitors (NH4Cl, Lys05) mildly enhanced the CQ- and BisGMA-induced cytotoxicity. These results indicate that DBAs stimulated cathepsin L, possibly due to their content of CQ and BisGMA that may induce cathepsin L in HDPCs. CQ and BisGMA stimulated lysosomal activity, autophagy, and apoptosis, possibly via induction of Beclin 1, ATG12, LC-3B, Bax, and p53 expression. In addition, CQ and BisGMA cytotoxicity was related to redox change and autophagy. These events are important role in pulpal changes after the restoration of tooth decay using CQ- and BisGMA-containing DBAs and resin composite.

Stimulation phosphatidylinositol 3-kinase/protein kinase B signaling by Porphyromonas gingivalis lipopolysacch aride mediates interleukin-6 and interleukin-8 mRNA/protein expression in pulpal inflammation.

BACKGROUND/PURPOSE: The signaling mechanisms for Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammation in human dental pulp cells are not fully clarified. This in vitro study aimed to evaluate the involvement of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in PgLPS-induced pulpal inflammation. METHODS: Human dental pulp cells (HDPCs) were challenged with PgLPS with or without pretreatment and coincubation with a PI3K/Akt inhibitor (LY294002). The gene or protein levels of PI3K, Akt, interleukin (IL)-6, IL-8, alkaline phosphatase (ALP), osteocalcin and osteonectin were analyzed by reverse transcription polymerase chain reaction (PCR), real-time PCR, western blotting, and immunofluorescent staining. In addition, an enzyme-linked immunosorbent assay was used to analyze IL-6 and IL-8 levels in culture medium. RESULTS: In response to 5 µg/ml PgLPS, IL-6, IL-8, and PI3K, but not Akt mRNA expression of HDPCs, was upregulated. IL-6, IL-8, PI3K, and p-Akt protein levels were stimulated by 10-50 µg/ml of PgLPS in HDPCs. PgLPS also induced IL-6 and IL-8 secretion at concentrations higher than 5 µg/ml. Pretreatment and co-incubation by LY294002 attenuated PgLPS-induced IL-6 and IL-8 mRNA expression in HDPCs. The mRNA expression of ALP, but not osteocalcin and osteonectin, was inhibited by higher concentrations of PgLPS in HDPCs. CONCLUSION: P. gingivalis contributes to pulpal inflammation in HDPCs by dysregulating PI3K/Akt signaling pathway to stimulate IL-6 and IL-8 mRNA/protein expression and secretion. These results are useful for understanding the pulpal inflammation and possible biomarkers of inflamed pulp diagnosis and treatment.

Emulating Clinical Diagnostic Reasoning for Jaw Cysts with Machine Learning.

The detection and classification of cystic lesions of the jaw is of high clinical relevance and represents a topic of interest in medical artificial intelligence research. The human clinical diagnostic reasoning process uses contextual information, including the spatial relation of the detected lesion to other anatomical structures, to establish a preliminary classification. Here, we aimed to emulate clinical diagnostic reasoning step by step by using a combined object detection and image segmentation approach on panoramic radiographs (OPGs). We used a multicenter training dataset of 855 OPGs (all positives) and an evaluation set of 384 OPGs (240 negatives). We further compared our models to an international human control group of ten dental professionals from seven countries. The object detection model achieved an average precision of 0.42 (intersection over union (IoU): 0.50, maximal detections: 100) and an average recall of 0.394 (IoU: 0.50-0.95, maximal detections: 100). The classification model achieved a sensitivity of 0.84 for odontogenic cysts and 0.56 for non-odontogenic cysts as well as a specificity of 0.59 for odontogenic cysts and 0.84 for non-odontogenic cysts (IoU: 0.30). The human control group achieved a sensitivity of 0.70 for odontogenic cysts, 0.44 for non-odontogenic cysts, and 0.56 for OPGs without cysts as well as a specificity of 0.62 for odontogenic cysts, 0.95 for non-odontogenic cysts, and 0.76 for OPGs without cysts. Taken together, our results show that a combined object detection and image segmentation approach is feasible in emulating the human clinical diagnostic reasoning process in classifying cystic lesions of the jaw.

Is periodontal disease a risk factor for oral cancer?

Aim This study aimed to investigate periodontal disease as a non-genetic risk factor for oral cancer.Design Case-control study.Patient population Two hundred patients, regardless of periodontal and adverse habits (smoking and alcohol) status, in the age group of 18-90 years were included in this institutional study. One hundred patients with histologically confirmed oral squamous cell carcinoma (OSCC) were included in the case group, while the control group had 100 patients without any oral cancer.Methods Multivariable examination to obtain socioeconomic and lifestyle risk factors was performed with a questionnaire for both the groups and compared statistically. Additionally, oral status (periodontal stage, clinical attachment loss, periodontal pocket depth, bleeding on probing, Silness-Loe plaque index, and decayed, missing, and filled teeth [DMFT] index) of both the groups was recorded and compared statistically.Results A significant correlation was found between age, gender and development of oral cancer. There was a significant co-relation between alcohol intake and oral cancer development. Surprisingly, there was no correlation between smoking habits and passive smoking with oral cancer development in the case group. Overall, 72.1% of case group patients had Stage 4 periodontitis, whereas 51.6% of control group patients had Stage 2 periodontitis. A significant correlation was found between the incidence of oral cancer and the stage of periodontitis.Conclusion The findings of the study support the hypothesis that periodontitis is an independent risk factor for oral cancer.

Metallothionein levels in gingival crevicular fluid, saliva, and serum of smokers and non-smokers with chronic periodontitis.

BACKGROUND: Metallothionein (MT), a cysteine rich protein is involved as a radical scavenger in several pathological conditions associated with oxidative stress; however, its role in periodontal disease still remains elusive. The aim of this cross-sectional study is to determine the serum, saliva and gingival crevicular fluid (GCF) levels of MT in smokers (S) and non-smokers (NS) with chronic periodontitis (CP), and compare them with those of periodontally healthy (PH) individuals. METHODS: A total of 85 participants were enrolled: 45 patients with CP (23 S [CP+S] and 22 NS [CP+NS]) and 40 PH individuals (20 S [PH+S] and 20 NS [PH+NS]). In all the study participants, clinical periodontal parameters (plaque index, gingival index, sulcus bleeding index, probing depth, and clinical attachment level) were recorded and samples of serum, saliva and GCF were collected. Enzyme-linked immunosorbent assay was used to determine the levels of MT in the samples. RESULTS: All periodontal clinical parameters were significantly higher in the CP groups as compared to PH groups (P < 0.05). MT levels in CP+S group were significantly raised in comparison to other three groups. There was no statistically significant difference in MT levels among CP+NS and PH+S groups (P > 0.05); however, relatively higher levels were observed in GCF and saliva in CP+NS group. When all the study groups were observed together, MT levels were positively correlated with clinical parameters. CONCLUSIONS: Results of present study suggest that smoking and CP can induce the synthesis of MT owing to increased oxidative stress and heavy metals intoxication. Further longitudinal studies with large sample size and an interventional arm are needed to substantiate the role of MT as a potential biomarker in periodontitis.

Prophylactic vaccination against periodontal disease: a systematic review of preclinical studies.

BACKGROUND: Periodontal vaccine against periodontopathic bacteria has been tested in various animal models, such as in non-human primates and murine species. The aim of this preliminary study is to systematically review all preclinical (i.e., animal) studies that present supporting evidence for the feasibility of formulating a prophylactic human periodontal vaccine. METHODS: A manual and electronic literature search was made for animal studies up to February 2010 that presented clinical, morphologic (alveolar bone level), and immunologic data for the efficacy of a prophylactic periodontal vaccine. A systematic approach was followed by two independent reviewers and included eligibility criteria for study inclusion, quality assessment, determination of outcome measures, screening method, data extraction, data synthesis, and drawing of conclusions. RESULTS: Only two randomized controlled animal trials qualified, and they reported exactly opposite effects of vaccine administration in Macaca fascicularis experimental periodontitis models by administration of two different agents (i.e., a negative effect with combined whole-cell antigens of Porphyromonas gingivalis and Prevotella intermedia in contrast to a positive effect with cysteine proteases of P. gingivalis). However, no statistical process could be applied to their results (data inadequately reported) in order to pool and evaluate the changes in outcome measures after vaccine administration, which, highlighted their mediocre study quality. CONCLUSIONS: Because of the insufficient quantity and quality of animal trials, no adequate evidence could be gathered to use the beneficial effects of these animal experiments to formulate a prophylactic human periodontal vaccine. Thus, good-quality animal trials are needed in this field of vaccine testing.